The present invention relates to nucleic acid primers and probes specific for organisms of the Mycobacterium avium complex (MAC) and to their use in nucleic acid amplification methods for the detection and differentiation of such organisms in biological samples. The invention also relates to diagnostic kits for detecting and differentiating the various organisms comprising the MAC.
The Mycobacterium avium complex (MAC) is composed of a large number of organisms, many of which are classified as M. avium or M. intracellulare. In addition, there are a number of organisms within the MAC that cannot be properly classified because they have the characteristics of both M. avium and M. intracellulare, or because they have combined characteristics of another mycobacterium with either M. avium or M. intracellulare (Wayne, et al., International J. Systematic Bacteriol., 43(3):482-489 (1993)).
The Mycobacterium avium complex consists of at least 26 serovars. These organisms were originally defined by their agglutination in the presence of specific agglutinating antisera (through immune reaction with their cell wall surface antigens). M. avium is considered to include serovars 1 through 6, 8 through 11, and 21, while M. intracellulare is considered to include serovars 7, 12-17, 19, 20, 25 (Wayne, et al., Clin. Microbiol. Rev. 5:1-25 (1992) and H. Saito, et al., J. Clin. Microbiol., 28:1694-1697 (1990)).
Frothingham et al. (J. Bacteriol., 175(10):2818-2825 (1993)) further classified these organisms into sequevars. The sequevar classification was derived by sequencing the 16s-23s rRNA internal transcribed spacer region of reference strains of organisms representing the MAC. This classification system, based on the genetic sequence, revealed a wide range of genetic diversity among non-M. avium, non-M. intracellulare MAC strains. Frothingham et al. classified these reference strains as Mav-A through Mav-D, for M. avium organisms, Min-A, for M. intracellulare organisms, and MAC-A through MAC-H, for all M. avium complex strains that fit in neither of the avium or intracellulare categories (see also, Frothingham, et al., J. Infect. Diseases, 169:305-312 (1994)).
Infections caused by members of the MAC have become a major clinical problem, particularly in individuals who have AIDS (especially those individuals with extremely low CD4 counts). Yakrus et al. (J. Clin. Microbiol., 28:926-929, (1990)) identified MAC organisms that were most frequently associated with disseminated disease: M. avium serovar 4 (40%), M. avium serovar 8 (17%), non-typeable MAC (13%), and M. avium serovar 1 (9%).
Laboratory diagnosis of disseminated MAC traditionally has been based on culture methodology. MAC culture methods are labor, material, and resource intensive, and require relatively long periods of time for definitive diagnosis. Because of this, a polymerase chain reaction (PCR) test for the detection of MAC infection would be advantageous.
PCR-based amplification of target nucleic acids allows rapid and sensitive detection of target DNA sequences. Amplified sequences accumulate to concentrations that are easily detected using non-isotopic detection methods. PCR technology theoretically allows the practitioner to identify a specific target nucleic acid in samples which may contain just a single target.
Kulski et al. (J. Clin. Microbiol., 33:668, (1995)) investigated the use of multiplex PCR to detect members of the Mycobacterium genus and to detect and differentiate M. tuberculosis, M. avium, and M. intracellulare. These investigators coamplified regions of the 16s rRNA gene with a region of the MPB70 gene to detect and differentiate M. avium, M. intracellulare, and M. tuberculosis. 
Abed et al. (Res. Microbiol., 146:405, 1995) amplified the entire 16s to 23s rRNA spacer region and used a secondary technique of RAPD fingerprinting to differentiate 56 strains belonging to 11 Mycobacterium species. Their PCR primers resided outside the intergenic region, with their forward and reverse primers targeted to the 16s rRNA gene and the 23s rRNA gene, respectively.
Barry et al. (EP Publication No. 0395292), and Rossau et al. (EP Publication No. 0525095), describe amplification of the entire 16s to 23s intergenic region, and diagnostic tests for bacterial organisms using probes targeted for sequences within the 16s to 23s intergenic region. As in Abed et al., the primers used by Barry et al. and Rossau et al. were targeted for sequences located within the genes encoding 16s or 23s rRNA.
Booth et al. (Infection and Immunity, 61(4):1509, (1993)) found a high degree of similarity between the 19 kd protein genes of M. avium, M. tuberculosis, and M. intracellulare. This high degree of similarity at the level of the gene persists at the protein level. Nair et al. (Molecular Microbiology, 6(11):1431, (1992)) demonstrated that the M. intracellulare gene encodes a seroactive lipoprotein. This lipoprotein was considered to be the M. intracellulare homologue of the serologically active 19 kd M. tuberculosis protein.
In designing assays to detect the presence of the MAC, the skilled artisan is faced with a significant challenge in selecting primer and probe combinations that detect all of the organisms that comprise the MAC and that do not cross react with non-MAC organisms. The present invention overcomes these problems by providing primers and probes specific for organisms of the MAC that can be used to co-amplify at least two, and preferably three, gene regions of these organisms.
In one embodiment, the present invention relates to a method for amplifying nucleic acids from MAC organisms. The method involves contacting a sample suspected of containing nucleic acids from MAC organisms with four different nucleoside triphosphates, a thermostable DNA polymerase, and a set of oligonucleotide primers specific for two or more of the following three gene regions: MacSequevar gene region, M. avium 19 kilodalton protein gene region, and M. intracellulare ribosomal protein sl gene region, under conditions such that the nucleic acids are amplified.
In another embodiment, the present invention relates to a method for amplifying and detecting nucleic acids from MAC organisms. The method involves contacting a sample suspected of containing nucleic acids from MAC organisms with four different nucleoside triphosphates, a thermostable DNA polymerase, and a set of oligonucleotide primers specific for two or more of the following three gene regions: MacSequevar gene region, M. avium 19 kilodalton protein gene region, and M. intracellulare ribosomal protein sl gene region, under conditions such that the nucleic acids are amplified. The amplified product is then denatured and detected.
In a further embodiment, the present invention relates to a method for detecting and distinguishing M. avium from other organisms of the Mycobacterium avium complex comprising contacting a sample suspected of containing nucleic acids from organisms of the MAC with an oligonucleotide probe comprising nucleotide sequence: 5xe2x80x2 CCC TGA GAC AAC ACT DGG TCC GTC C 3xe2x80x2 (SEQ ID NO:1), wherein D is any nucleotide other than C and detecting the presence or absence of a complex formed between the probe and nucleic acids present in the sample.
Various other objects and advantages of the present invention will be apparent from the detailed description of the invention.
All publications mentioned herein are hereby incorporated by reference.
The development of a nucleic acid co-amplification system that detects all MAC organisms without crossreacting with non-MAC mycobacteria is very challenging. Applicants have overcome several obstacles to arrive at the present invention relating to the amplification and/or detection of nucleic acids from MAC organisms.
In the present invention, three gene regions have been identified that are compatible with each other in a multiplexed system and can, therefore, be used in co-amplification assays to amplify nucleic acids from MAC organisms. These three gene regions are the MacSequevar region (Msqv) found in M. avium, M. intracellulare, and non-M. avium, non-M. intracellulare MAC organisms, the M. avium 19 kilodalton protein (MAV19k), and the M. intracellulare ribosomal protein sl gene (rpsl). Using primers specific for two or more of these regions, amplification can be carried out in a multiplexed fashion in the presence of an internal positive control (IPC) that allows detection of false negative results due to problems in sample preparation, amplification and/or detection.
The present invention relates to methods for amplifying nucleic acids from MAC organisms. In the methods of the present invention, specific gene regions of the MAC genome are amplified by contacting a biological sample suspected of containing nucleic acids of such organisms with primer sets specific for two or more of the three gene regions. Preferably primer sets specific for all three gene regions are used to co-amplify the three regions. In addition to the primers, the biological sample is also contacted with PCR reagents, such as four different nucleoside triphosphates and a thermostable DNA polymerase, under conditions such that any MAC organism present in the sample will have its target nucleic acid amplified. Examples of primers suitable for use in the present invention include, but are not limited to, those shown below in Table 1.
Other primer sets specific for the three gene regions of interest could be readily determined by those skilled in the art.
Once the nucleic acids of the MAC are amplified, the presence or absence of the amplified target nucleic acids can be detected using known detection methods. For example, the amplified target nucleic acid can be detected using oligonucleotide probes specific for the amplified gene regions. Those skilled in the art can readily identify oligonucleotide probes that would be suitable to detect the amplified gene regions given the primer sets used. Oligonucleotide probes suitable for use in the present invention include, but are not limited to the oligonucleotides set forth in Table 1.
The present invention also relates to methods of detecting and distinguishing M. avium from other organisms of the MAC. This is achieved by contacting a sample suspected of containing nucleic acids of MAC organisms with a probe comprising the nucleotide sequence:
5xe2x80x2 CCC TGA GAC AAC ACT DGG TCC GTC C 3xe2x80x2 (SEQ ID NO:1) ,
wherein D is any nucleotide other than C. Preferably, D is G or U. Such probes are specific to the 16s to 23s rRNA intergenic region and are M. avium specific. The single base change from C to any other nucleotide alters the specificity of the probe making it highly specific for M. avium. 
The general principles and conditions for amplification and detection of nucleic acids using polymerase chain reaction (PCR) are quite well known, the details of which are provided in numerous references including U.S. Pat. Nos 4,683,195 (Mullis et al.), 4,683,202 (Mullis), and 4,965,188 (Mullis et al.), all of which are incorporated herein by reference. Thus, in view of the teaching in the art and the specific teaching provided herein, a worker skilled in the art should have no difficulty in practicing the present invention by co-amplifying two or more gene regions of organisms of the MAC to detect disseminated M. avium complex.
The term xe2x80x9coligonucleotidexe2x80x9d refers to a molecule comprised of one or more deoxyribonucleotides or ribonucleotides, such as primers, probes, and nucleic acid fragments to be detected.
The term xe2x80x9cprimerxe2x80x9d refers to an oligonucleotide, whether naturally occurring or synthetically produced, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand (that is, template) is induced, such conditions include the presence of other PCR reagents, and suitable temperature and pH.
The primer is preferably single stranded for maximum efficiency in amplification, but can contain a double stranded region if desired. It must be long enough to prime the synthesis of extension products in the presence of the DNA polymerase. The exact size of each primer will vary depending upon the use contemplated, the concentration and sequence of the primer, the complexity of the targeted sequence, the reaction temperature, and the source of the primer. Generally, the primers used in this invention will have from 12 to 60 nucleotides, and preferably, they have from 16 to 40 nucleotides. More preferably, each primer has from 18 to 35 nucleotides.
Primers useful herein can be prepared using known techniques and equipment, including for example an ABI DNA Synthesizer (available from Applied Biosystems) or a Biosearch 8600 Series or 8800 Series Synthesizer (available from Milligen-Biosearch, Inc.). Procedures for using this equipment are well known and described for example in U.S. Pat. No. 4,965,188 (Gelfand et al.), incorporated herein by reference. Naturally occurring primers isolated from biological sources may also be useful (such as restriction endonuclease digests).
As used herein, a xe2x80x9cprobexe2x80x9d is an oligonucleotide which is substantially complementary to a nucleic acid sequence of the target nucleic acid and which is used for detection or capture of the amplified target nucleic acid.
In the present invention, sequence specific primers and probes are provided. It will be apparent to those skilled in the art that additional sequence specific primers and probes can be prepared by, for example, the addition of nucleotides to either the 5xe2x80x2 or 3xe2x80x2 ends, which nucleotides are complementary or noncomplementary to the target sequence. Such compositions are within the scope of this invention.
The primers and/or the probes used in the present invention can, optionally, be labeled. Using known methods in the art, the primers and/or probes can be labeled with a specific binding ligand (such as biotin), an enzyme (such as glucose oxidase, peroxidases, uricase, and alkaline phosphatase), radioisotopes, electron-dense reagents, chromogens, fluorogens, phosphorescent moieties or ferritin. Preferably, the label is a specific binding ligand. More preferably, the label is biotin or a derivative thereof, streptavidin or a derivative thereof or a hapten.
A xe2x80x9cPCR reagentxe2x80x9d refers to any of the reagents considered essential for PCR, namely a set of primers for each target nucleic acid, a DNA polymerase (preferably a thermostable DNA polymerase), a DNA polymerase cofactor, and one or more deoxyribonucleoside-5xe2x80x2-triphosphates (dNTP""s). Other optional reagents and materials used in PCR are described below. These reagents can be provided individually, as part of a test kit, or in reagent chambers of test devices.
A DNA polymerase is an enzyme that will add deoxynucleoside monophosphate molecules to the 3xe2x80x2-hydroxy end of the primer in a complex of primer and template, but this addition is in a template dependent manner. Generally, synthesis of extension products proceeds in the 5xe2x80x2 to 3xe2x80x2 direction of the newly synthesized strand until synthesis is terminated. Useful DNA polymerases include, for example, E. coli DNA polymerase I, T4 DNA polymerase, Klenow polymerase, reverse transcriptase and others known in the art. Preferably, the DNA polymerase is thermostable meaning that it is stable to heat and preferentially active at higher temperatures, especially the high temperatures used for priming and extension of DNA strands. More particularly, thermostable DNA polymerases are not substantially inactive at the high temperatures used in polymerase chain reactions as described herein. Such temperatures will vary depending on a number of reaction conditions, including pH, nucleotide composition, length of primers, salt concentration and other conditions known in the art.
A number of thermostable DNA polymerases have been reported in the art, including those mentioned in detail in U.S. Pat. Nos. 4,965,188 (Gelfand et al.) and 4,889,818 (Gelfand et al.), both incorporated herein by reference. Particularly useful polymerases are those obtained from various Thermus bacterial species, such as Thermus aquaticus, Thermus thermophilus, Thermus filiformis, and Thermus flavus. Other useful thermostable polymerases are obtained from various microbial sources including Thermococcus literalis, Pyrococcus furiosus, Thermotoga sp. and those described in WO-A-89/06691 (published Jul. 27, 1989). Some useful thermostable polymerases are commercially available, such as, AmpliTaq(copyright), Tth, and UlTma(copyright) from Perkin Elmer, Pfu from Stratagene, and Vent and Deep-Vent from New England Biolabs. A number of techniques are also known for isolating naturally-occurring polymerases from organisms, and for producing genetically engineered enzymes using recombinant techniques.
A DNA polymerase cofactor refers to a nonprotein compound on which the enzyme depends for activity. Thus, the enzyme is catalytically inactive without the presence of cofactor. A number of materials are known cofactors including, but not limited to, manganese and magnesium salts, such as chlorides, sulfates, acetates and fatty acids salts. Magnesium chlorides and sulfates are preferred.
Also needed for PCR are two or more deoxyribonucleoside-5xe2x80x2-triphosphates, such as two or more of dATP, dCTP, dGTP, dTTP and dUTP. Analogues such as dITP and 7-deaza-dGTP are also useful. Preferably, the four common triphosphates (dATP, dCTP, dGTP and dTTP) are used together.
The PCR reagents described herein are provided and used in PCR in suitable concentrations to provide amplification of the target nucleic acid. The minimal amounts of primers, DNA polymerase, cofactors and deoxyribonucleoside-5xe2x80x2-triphosphates needed for amplification and suitable ranges of each are well known in the art. The minimal amount of DNA polymerase is generally at least about 0.5 units/100 xcexcl of solution, with from about 2 to about 25 units/100 xcexcl of solution being preferred, and from about 7 to about 20 units/100 xcexcl of solution being more preferred. Other amounts may be useful for given amplification systems. A xe2x80x9cunitxe2x80x9d is defined herein as the amount of enzyme activity required to incorporate 10 nmoles of total nucleotides (dNTP""s) into an extending nucleic acid chain in 30 minutes at 74xc2x0 C. The minimal amount of primer is at least about 0.075 xcexcmolar with from about 0.1 to about 2 xcexcmolar being preferred, but other amounts are well known in the art. The cofactor is generally present in an amount of from about 2 to about 15 mmolar. The amount of each dNTP is generally from about 0.25 to about 3.5 mmolar.
The PCR reagents can be supplied individually, or in various combinations, or all in a buffered solution having a pH in the range of from about 7 to about 9, using any suitable buffer, many of which are known in the art.
Other reagents that can be used in PCR include, for example, antibodies specific for the thermostable DNA polymerase. Antibodies can be used to inhibit the polymerase prior to amplification. Antibodies useful in the present invention are specific for the thermostable DNA polymerase, inhibit the enzymatic activity of the DNA polymerase at temperatures below about 50xc2x0 C., and are deactivated at higher temperatures. Useful antibodies include, monoclonal antibodies, polyclonal antibodies and antibody fragments. Preferably, the antibody is monoclonal. The antibodies useful in the present invention can be prepared using known methods such as those described in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y. (1988).
Representative monoclonal antibodies are described in U.S. Pat. No. 5,338,671 (Scalice et al.), the contents of which are hereby incorporated by reference. Two such monoclonal antibodies are readily obtained by a skilled artisan using conventional procedures, and starting materials including either of hybridoma cell lines HB 11126 or 11127, deposited with the American Type Culture Collection (ATCC) (Rockville, Md.). The monoclonal antibody is present in an amount of from about 5:1 to about 500:1 molar ratio to the DNA polymerase.
A target nucleic acid, including that from a MAC organism, can be obtained from any of a variety of sources such as peripheral blood mononuclear cells (PBMC""s), whole blood, respiratory fluids, lymph, and stool. Generally, it is extracted in some conventional manner to make it available for contact with the primers and other PCR reagents. If the target nucleic acid is double stranded, the two strands must be separated before priming can occur. Denaturation can be accomplished using any of the known techniques such as heat treatment, physical treatment or chemical treatment.
Amplification is preferably conducted in a continuous, automated manner so that the reaction mixture is temperature cycled in a controlled manner for desired preset times. A number of instruments have been developed for this purpose and are available to those skilled in the art. Preferably, amplification is carried out in a closed reaction vessel, such as the chemical test pack described in U.S. Pat. No. 5,229,297, which vessel is processed on the instrument described in U.S. Pat. No. 5,089,233.
Amplified nucleic acids can be detected in a number of known ways, such as those described in U.S. Pat. No. 4,965,188 (Gelfand et al.). For example, the amplified nucleic acids can be detected using Southern blotting, dot blot techniques, or nonisotopic oligonucleotide capture detection with a labeled probe. Alternatively, amplification can be carried out using primers that are appropriately labeled, and the amplified primer extension products can be detected using procedures and equipment for detection of the label.
In a preferred embodiment, the amplified target nucleic acid is detected using an oligonucleotide probe that is labeled for detection and can be directly or indirectly hybridized with the amplified target. The probe may be soluble or attached to a solid support. When multiple probes are used in the present invention, the probes can be attached to a solid support at different locations. Alternatively, the probes can be attached to the solid support as a mixture at the same location. In another preferred embodiment, one or more of the primers used to amplify the target nucleic acid is labeled, for example, with a specific binding moiety. The resulting primer extension product into which the labeled primer has been incorporated can be captured with a probe. Detection of the amplified target hybridized to the probe can be achieved by detecting the presence of the labeled probe or labeled amplified target using suitable detection equipment and procedures that are well known in the art. Certain labels may be visible to the eye without the use of detection equipment.
In a more preferred embodiment, one or more of the primers used to amplify the target nucleic acid is labeled with biotin and the biotinylated amplified target nucleic acids are hybridized to probes attached to a solid support. The bound targets are then detected by contacting them with a streptavidin-peroxidase conjugate in the presence of an oxidant, such as hydrogen peroxide, and a suitable dye-forming composition. For example, useful dye-providing reagents include tetramethylbenzidine and derivatives thereof, and leuco dyes, such as triarylimidazole leuco dyes as described in U.S. Pat. No. 4,089,747 (Bruschi).
Preferably, amplification and detection are carried out in a closed reaction vessel to reduce the risk of contamination. Both amplification and detection can be carried out in a closed reaction vessel as described in U.S. Pat. No. 5,229,297, without opening up the reaction vessel during the process.
As used herein, when in reference to time the term xe2x80x9caboutxe2x80x9d refers to +/xe2x88x9210% of that time limit. When used in reference to temperatures, the term xe2x80x9caboutxe2x80x9d refers to +/xe2x88x925xc2x0 C.
The following Examples are provided to illustrate certain embodiments of the present invention, and are not to be construed as limiting the invention.